The catalytic action of aminoacyl-tRNA synthetases consists of two partial reactions: 1. amino acid plus enzyme enzyme-aminoacyl-AMP plus PP. 2. Enzyme aminoacyl-AMP plus tRNA aminoacyl-tRNA plus AMP plus enzyme. The roles of specific amino acid residues in the catalytic process will be investigated. One approach will be to use site-specific reagents for linking substrates to the enzyme. This approach will use known reagents and new compounds that are analogues of the amino acid, ATP and tRNA to probe the areas of the protein to which each substrate is bound. Enzyme modified by each reagent will be assayed for the partial reactions characteristic of these catalysts and will also be analyzed chemically to determine the number of chemical modifications and the points of attachment. The partial reactions will be assayed using exogenous aminoacyl-adenylate as substrate for both pyrophosphorolysis (reaction 1) and transfer (reaction 2) in order to determine the properties related to single-step reactions. A second approach will be to use reagents specific for amino acid side chains. In this case mild conditions will be used to distinguish the residues that react most rapidly and to correlate modification of the protein with alterations in catalytic activity, again, distinguishing effects on the partial reactions. Reagents that inactivate will be tested in the presence and absence of substrates; when protection by a substrate is detected, isotopic chromophoric reagent will be used in a second step to identify the critical group(s) in the enzyme. In addition attempts will be continued to obtain crystals and start an analysis of the three-dimensional structure by x-ray crystallography. All of the preceeding analyses give static information. It is proposed to determine spacial relationships and changes in the conformation of the enzyme by using spin-labeled substrate analogues for ESR analyses.